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ObjectiveTo investigate the diagnostic and prognostic role of gastric fluid DNA (gfDNA) in gasric cancer (GC) patients and controls submitted to upper digestive endoscopy.DesignThe concentration of gfDNA was evaluated in 941 samples, including subjects with normal gastric mucosa (n = 10), peptic diseases (n = 596), pre-neoplastic conditions (n = 99), and cancer (n = 236). gfDNA levels were evaluated according to age, gender, BMI, gastric fluid’s pH, use of proton-pump inhibitors, GC tumor subtypes, histological grades, clinical stages, and disease progression/outcome.ResultsIn the non-cancer group, we observed that gfDNA levels are increased in women as compared to men (p=7.44e-4). Remarkably, gfDNA levels are increased in GC patients as compared to non-GC (normal + peptic diseases, p=5.67e-13) and in GC versus pre-neoplastic disease (p=1.53e-6). Similar differences were also seen when more advanced tumors (T3) were compared to early stages (T2 and below) (p=5.97-4). Moreover, our results suggest the prognostic value of gfDNA as GC-patients with higher gfDNA concentrations (-3), which parallels with better disease-free survival (p= 0.014).ConclusionThese findings highlight the significance of collecting and studying stomach fluids from gastric cancer patients and reveals the potential impact of this approach as well as its diagnostic and prognostic value for disease management.What is already known on this topic-The DNA contained in biofluids that are in direct contact with tumor lesions is a valuable source of information for cancer diagnosis treatment.-Gastric fluids are in direct contact with gastric cancer (GC) lesions and immune cell infiltrates and may be a reliable source of biomarkers with multiple applications.-Liquid biopsies are usually based on the detection and analysis of cell-free DNA found in the plasma, urine or saliva.-Studies of the DNA present in the gastric fluids of patients (gfDNA), collected during routine endoscopy, are scarce and limited to assessing mutation profiles. It’s diagnostic and prognostic potential has not been studied so far.What this study adds-An analysis of gfDNA concentrations in 941 samples revealed increased DNA levels in GC patients as compared to non-GC individuals.-When tumor stages were compared, gfDNA levels are elevated in more advanced disease.-For GC subjects, increased gfDNA was positively associated with immune cell infiltrates and reduced tumor recurrence, suggesting this to be an informative and very low-cost prognostic marker.How this study might affect research, practice or policy-gfDNA collected during routine upper digestive diagnostic endoscopy can be used as an additional and very simple biomarker useful during patient follow-up and to determine cancer prognosis.-In the future gfDNA assessment has the potential to contribute to a range of applications, such as monitoring treatment response, indicating immune cell infiltration and may potentially indicate cases with progressive disease non-responsive to chemotherapy.

About 4% to 7% of the non–small-cell lung cancer patients have anaplastic lymphoma kinase (ALK) rearrangements, and specific targeted therapies improve patients' outcomes significantly. ALK gene fusions are detected by immunohistochemistry or fluorescent in situ hybridization as gold standards in South America. Next-generation sequencing–based assays are a reliable alternative, able to perform simultaneous detection of multiple events from a single sample. We analyzed 4240 non–small-cell lung cancer samples collected in 37 hospitals from Chile, Brazil, and Peru, where ALK rearrangements were determined as part of their standard of care (SofC) using either immunohistochemistry or fluorescent in situ hybridization. A subset of 1450 samples was sequenced with the Oncomine Focus Assay (OFA), and the concordance with the SofC tests was measured. An orthogonal analysis was performed using a real-time quantitative PCR echinoderm microtubule-associated protein-like 4-ALK fusion detection kit. ALK fusion prevalence is similar for Chile (3.67%; N = 2142), Brazil (4.05%; N = 1013), and Peru (4.59%; N = 675). Although a comparison between OFA and SofC assays showed similar sensitivity, OFA had significantly higher specificity and higher positive predictive value, which opens new opportunities for a more specific determination of ALK gene rearrangements.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Programa Nacional de Apoio à Atenção Oncológica (Pronon) Associacao Beneficiente Alzira Denise Hertzog Silva (ABADHS) Sporadic and inflammatory forms of colorectal cancer (CRC) account for more than 80% of cases. Recent publications have shown mechanistic evidence for the involvement of gut bacteria in the development of both CRC-forms. Whereas, colon and rectal cancer have been routinely studied together as CRC, increasing evidence show these to be distinct diseases. Also, the common use of fecal samples to study microbial communities may reflect disease state but possibly not the tumor microenvironment. We performed this study to evaluate differences in bacterial communities found in tissue samples of 18 rectal-cancer subjects when compared to 18 non-cancer controls. Samples were collected during exploratory colonoscopy (non-cancer group) or during surgery for tumor excision (rectal-cancer group). High throughput 16S rRNA amplicon sequencing of the V4V5 region was conducted on the Ion PGM platform, reads were filtered using Qiime and clustered using UPARSE. We observed significant increases in species richness and diversity in rectal cancer samples, evidenced by the total number of OTUs and the Shannon and Simpson indexes. Enterotyping analysis divided our cohort into two groups, with the majority of rectal cancer samples clustering into one enterotype, characterized by a greater abundance of Bacteroides and Dorea. At the phylum level, rectal-cancer samples had increased abundance of candidate phylum OD1 (also known as Parcubacteria) whilst non-cancer samples had increased abundance of Planctomycetes. At the genera level, rectal-cancer samples had higher abundances of Bacteroides, Phascolarctobacterium, Parabacteroides, Desulfovibrio, and Odoribacter whereas non-cancer samples had higher abundances of Pseudomonas, Escherichia, Acinetobacter, Lactobacillus, and Bacillus. Two Bacteroides fragilis OTUs were more abundant among rectal-cancer patients seen through 16S rRNA amplicon sequencing, whose presence was confirmed by immunohistochemistry and enrichment verified by digital droplet PCR. Our findings point to increased bacterial richness and diversity in rectal cancer, along with several differences in microbial community composition. Our work is the first to present evidence for a possible role of bacteria such as B. fragilis and the phylum Parcubacteria in rectal cancer, emphasizing the need to study tissue-associated bacteria and specific regions of the gastrointestinal tract in order to better understand the possible links between the microbiota and rectal cancer. CIPE AC Camargo Canc Ctr, Med Genom Lab, Sao Paulo, Brazil Univ Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, Brazil Univ Sao Paulo, Cursode Posgrad Bioinformat, Sao Paulo, Brazil AC Camargo Canc Ctr, Dept Pelv Surg, Sao Paulo, Brazil AC Camargo Canc Ctr, Dept Pathol, Sao Paulo, Brazil Univ Fed Sao Paulo, Coll Med, Dept Gynecol, Lab Mol Gynecol, Sao Paulo, Brazil Hosp Sirio Libane, Ctr Oncol Mol, Sao Paulo, Brazil Univ Sao Paulo, Food Res Ctr FoRC, Fac Ciencias Farmaceut, Dept Alimentos & Nutr Expt, Sao Paulo, Brazil AC Camargo Canc Ctr, Dept Clin Oncol, Sao Paulo, Brazil AC Camargo Canc Ctr, Lab Computat Biol & Boinformat, Sao Paulo, Brazil Virginia Tech, Biocomplex Inst, Blacksburg, VA USA Univ Sao Paulo, Fac Med, Inst Psychiat, Lab Neurosci LIM Alzira Denise Hertzog Silva 27, Sao Paulo, Brazil Laboratory of Molecular Gynecology, Department of Gynecology, Medicine College, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil FAPESP: 2015/01507-7 FAPESP: 2013/07914-8 CAPES: 88887.062078/2014-00 CAPES: 3385/2013 PRONON: 25000.055.167/2015-23 Web of Science

O melanoma é classificado como o tipo mais agressivo do câncer de pele. A terapia alvodirigida combinada, com inibidores de BRAF e MEK, considerada a opção terapêutica padrão para o tratamento de pacientes em estágios avançados que possuem mutação em BRAF, resulta em uma taxa de sobrevida em 5 anos de apenas 20%. A transferência de informações via Vesículas Extracelulares (VEs) tem sido descrita como um dos mecanismos capazes de modular os processos de resistência e progressão maligna. No melanoma, a presença de moléculas antitumorais em VEs está associada a maiores taxas de resposta ao tratamento e melhores prognósticos. O miR-195, considerado um supressor tumoral, é down-regulado em melanomas e sua superexpressão resulta na diminuição da proliferação celular e aumento da sensibilidade à quimioterapia com agentes alquilantes e inibidores de BRAF. Neste sentido, o objetivo deste trabalho foi avaliar o efeito da superexpressão de miR-195 na carga de VEs liberadas por células de melanoma e o papel destas partículas na regulação da sensibilidade de células naive a terapia alvo-dirigida combinada. Diferentes linhagens de melanoma humano (A375, SKMel-05, SKMel-28 e UACC-62) foram transfectadas com o miR-195, resultando no aumento da concentração de VEs liberadas, as quais apresentaram menor tamanho em relação ao controle. Análises de expressão gênica por RT-qPCR mostraram que a transfecção de miR-195 induz o aumento da expressão de GTPases envolvidas na biogênese de exosomos, enquanto genes envolvidos na produção de microvesículas têm sua expressão diminuída. VEs secretadas por células transfectadas com este miRNA foram capazes de transferir seu efeito citostático, induzindo a expressão de genes antiproliferativos, como CDKN1A e CDKN3, e diminuindo a Página | 2 expressão de genes pró-proliferativos, como CCND1 e YAP1, em células naive. VEs contendo miR-195 também impactaram o crescimento tumoral in vivo, diminuindo a cinética de crescimento dos tumores. Além disso, o tratamento com VEs miR-195 foi capaz de aumentar a sensibilidade de células naive a terapia alvo-dirigida combinada, resultando em maior expressão de BAD, menor expressão de BCL2L1 e consequentemente maior porcentagem de morte celular, além da diminuição do potencial clonogênico das células. Adicionalmente, com relação ao mecanismo de regulação de miR-195, dados do TCGA mostraram maiores níveis de metilação deste miRNA em melanomas em relação a tecidos saudáveis, entretanto, sua expressão foi restaurada após o tratamento das células com Azacitidina em apenas uma das três linhagens testadas. Análises in silico mostraram que diversos lncRNAs capazes de inibir miR195 encontram-se superexpressos em melanoma, representando outro possível mecanismo de down-regulação. Em resumo, a superexpressão de miR-195 representa uma estratégia promissora para sensibilizar células de melanoma a terapia via transferência horizontal de informação Melanoma is considered the most aggressive form of skin cancer. Combined targeted therapy, with BRAF and MEK inhibitors, considered the standard treatment for BRAF-mutated metastatic melanoma patients, results in 5 years overall survival of 20%. Extracellular Vesicles (EVs)-mediated transfer of information has been reported as a mechanism capable of regulating tumor resistance and progress. The presence of antitumoral molecules within EVs is related to increased response rates and better prognosis for melanoma patients. MiR-195, considered a tumor suppressor miRNA, is down-regulated in melanoma and its overexpression results in decreased cells proliferation and increased sensitivity to chemotherapy with alkylating agents and BRAF inhibitors. We aimed to analyze whether miR-195 overexpression could modulate EVs cargo and the role of these particles in naïve cells response to combined targeted treatment. A375, SKMel-5, SKMel-28 and UACC-62 cells were transfected with miR-195, resulting in increased EVs release and decreased EVs size. Gene expression analysis by RT-qPCR showed that miR-195 mimic induces the expression of GTAPases involved in exosomes biogenesis, while genes involved in microvesicles shedding are down-regulated upon transfection. EVs derived from cells overexpressing miR-195 could transfer its cytostatic effect, inducing the expression of anti-proliferative genes, such as CDKN1A and CDKN3, and inhibiting proproliferative genes, including CCND1 and YAP1, in naïve cells. EVs containing miR-195 also impaired in vivo tumor growth, reducing tumors growth kinetics. Treatment with miR-195 EVs could also sensitize naive cells to combined targeted therapy, resulting in higher expression of BAD, inhibition of BCL2L1 and consequently increased cell death, in addition to decreased Página | 4 clonogenic potential. Additionally, regarding the mechanisms of miR-195 regulation, TCGA data showed higher levels of methylation of this miRNA in melanoma tissues compared to controls, nevertheless, treatment of melanoma cells with Azacitidine only restored miR-195 expression in one out of three cell lines. In silico analysis showed that several lncRNAs capable to sponge miR-195 are overexpressed in melanomas, representing another possible mechanism of down-regulation. In summary, miR-195 overexpression represents a promising strategy to sensitize melanoma cells through horizontal transfer of information

O lincRNA PVT1 (Plasmacytoma Variant Translocation 1) é um RNA longo não codificador de proteínas (ncRNA) descrito como um oncogene sendo superexpresso em vários tipos de cânceres. LincRNA PVT1 está localizado na região genômica 8q24, também conhecida como \'gene desert\'. O nível de expressão do lincRNA PVT1 está associado ao aumento do risco de câncer de próstata (PCa) e está correlacionado com os níveis de expressão do receptor de andrógeno (AR). No entanto, o mecanismo do envolvimento do lincRNA PVT1 com o AR no desenvolvimento de câncer de próstata ainda não está bem esclarecido. Aqui, nós testamos a hipótese que a formação do complexo AR-EZH2-PVT1 participa na regulação da expressão gênica em câncer de próstata, nas células LNCaP. A imunoprecipitação de ribonucleoproteínas seguida de PCR quantitativo (RIP-qPCR) revelou que o lincRNA PVT1 está associado fisicamente ao AR (12% do input) e à metiltransferase EZH2, proteína componente do complexo repressor Polycomb 2 (36% do input) sob condições suplementadas com andrógeno (+R1881). O lincRNA PVT1 também está associado fisicamente ao AR (10% de input) e à EZH2 (42% de input) em condições de privação de andrógeno (-R1881). Assim, a associação física entre lincRNA PVT1, AR e EZH2 é independente do hormônio andrógeno. Usando uma abordagem de estudo em larga-escala de perda e ganho de função, nossos resultados mostraram que o silenciamento do lincRNA PVT1 em células LNCaP na presença de andrógeno restaura a expressão parcialmente, totalmente ou causa superexpressão de 160 genes que tiveram a expressão inibida por andrógeno. Entre esses genes, destacamos genes envolvidos na regulação da diferenciação celular, em componentes da junção célula-célula, na inibição da migração e invasão celular e no desencadeamento da via apoptótica. Imunoprecipitação da cromatina seguida de PCR quantitativo (ChIP-qPCR), em cultura de células LNCaP suplementada com andrógeno sob silenciamento do lincRNA PVT1, mostrou aumento significativo na ocupação pela marca de histona ativadora H3K27Ac do promotor do gene NOV, um dos genes que tiveram sua expressão aumentada com o silenciamento de PVT1. O ChIP-qPCR também mostrou, após o silenciamento do lincRNA PVT1, um aumento significativo da marca H3K27me3 na região enhancer do gene NOV, uma característica de enhancers poised (prontos para ativação). Em conclusão, nós fornecemos a primeira evidência experimental para um mecanismo de ação do oncogene lincRNA PVT1 em células de câncer de próstata e demonstramos que sua ação inibidora da expressão afeta genes alvo que facilitam a proliferação e migração de células do câncer de próstata, sugerindo que o lincRNA PVT1 é um novo agente no complexo mecanismo de repressão transcricional envolvendo um RNA silenciador, o receptor de andrógeno (AR) e o potenciador de Zeste homólogo 2 (EZH2) no remodelamento da cromatina em células LNCaP. Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) is an oncogene known to be overexpressed in various types of cancer. PVT1 lincRNA is located in the wellknown cancer-related genomic region 8q24, also known as \'gene desert. PVT1 lincRNA level of expression is associated with increased prostate cancer (PCa) risk and is correlated with androgen receptor (AR) expression levels. However, the mechanism of PVT1 and AR involvement in the development of prostate cancer is still unclear. Here, we tested the hypothesis that formation of the complex AR-EZH2-PVT1 participates in the regulation of gene expression in prostate cancer, in LNCaP cells. Ribonucleoprotein immunoprecipitation followed by quantitative PCR (RIP-qPCR) revealed that PVT1 lincRNA binds both the AR (12 % of PVT1 input) and the methyltransferase EZH2 from the Polycomb repressive complex 2 (36 % of input) under androgen-supplemented conditions (+R1881). PVT1 also binds both AR (10 % of input) and EZH2 (42 % of input) under androgen-deprived conditions (-R1881). Thus, PVT1 binding to AR and EZH2 is independent of the androgen hormone. Using a large-scale loss and gain of function approach, our results show that PVT1 knockdown (KD) in LNCaP in the presence of androgen restores the expression partially, fully or causes overexpression of 160 genes that are inhibited by androgen. Among these genes, we highlight genes involved in regulation of cell differentiation, in components of cell-cell junction, in inhibition of cell migration and invasion and in triggering of the apoptotic pathway. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) with LNCaP cells in androgen-supplemented cultures under PVT1 lincRNA knockdown showed a significant increase in occupancy by the histone activation mark H3K27Ac of the promoter region of the NOV gene, one of the genes that had an increased expression upon PVT1 silencing. ChIPqPCR also showed a significant increase upon PVT1 lincRNA silencing of the H3K27me3 histone mark in the enhancer region of the NOV gene, a distinct feature of poised enhancers. In conclusion, we provide first experimental evidence for a mechanism of action of PVT1 lincRNA oncogene in prostate cancer cells, and show that its inhibitory action affects targetgenes that facilitate proliferation and migration of prostate cancer cells, thus suggesting PVT1 lincRNA as a novel lncRNA player in the complex mechanism of transcriptional repression involving a silencer RNA, the androgen receptor (AR) and the Enhancer of zeste homolog 2 (EZH2) in chromatin remodeling in LNCaP cells.

CSCs, caracterizadas pela alta atividade da ALDH1 e expressão (ou não) de marcadores de células-tronco embrionárias OCT-4, NANOG, SOX2, KLF4, LIN2, estão presentes em tumores de mama HER2+. Mamosferas são estruturas celulares esferóides in vitro enriquecidas em CSCs. Neste trabalho, buscou-se um melhor entendimento do papel dos miRNAs -221, -222 e -4728-3p na biologia das CSCs. Foi observado alto percentual de células ALDH1+ (citometria) em mamosferas das linhagens MCF-7, SKBR3 e BT-474; alta expressão (qRT-PCR) de miR-221 e -222 em mamosferas da linhagem MCF-7 e de tumores de pacientes, além de alta expressão de HER2 em mamosferas das linhagens MCF-7 e SKBR3. A MFE de células MCF-7 mostrou-se aumentada após a indução (com lentivetores) da superexpressão dos miRNAs -221 e -222. A resistência ao quimioterápico paclitaxel estava aumentada em mamosferas que superexpressavam o miRNA-222. A superexpressão de miR-4728-3p, localizado em um intron de HER2, levou a um aumento das subpopulações ALDH1+ em duas linhagens celulares reforçando seu envolvimento com a biologia de CSCs. CSCs, characterized by high activity of ALDH1 and high (or not) expression of embryonic stem cell markers OCT-4, NANOG, SOX2, KLF4, LIN28 are present in tumors with HER2 amplification. Mammospheres are spheroid cell structures in vitro enriched by CSCs. In this study, we aim to contribute to the better understanding the role of miRNAs -221, 222, 4728-3p in the the biology of CSCs. We observed high percentage of ALDH+ cells (cytometry) in mammospheres of MCF-7, SKBR3 and BT-474 cell lines; high expression (qRT-PCR) of miR-221 and -222 in mammospheres of MCF-7 cell line and cells derived from patients, moreover HER2 is upregulated in mammospheres from MCF-7 and SKBR3 cell lines. MFE is higher in MCF-7 cell line after induction (with lentivectors) of miR-221 and -222 expression. The resistance to paclitaxel was increased in mammopheres that were overexpressing miRNA-222. Overexpression of miR-4728-3p, located in an intron of HER2 gene, induced the increment of ALDH1+ subpopulations in two cell lines, reinforcing its role in the biology of CSCs.